Jiang L et al. (2022), Frequent SLC12A3 mutations in Chinese Gitelman ...

XB-ART-58695

Endocr Connect 2022 Jan 27;111:. doi: 10.1530/EC-21-0262.

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Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder.

Jiang L , Peng X , Zhao B , Zhang L , Xu L , Li X , Nie M , Chen L .

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Purposes: This study was conducted to identify the frequent mutations from reported Chinese Gitelman syndrome (GS) patients, to predict the three-dimensional structure change of human Na-Cl co-transporter (hNCC), and to test the activity of these mutations and some novel mutations in vitro and in vivo. Methods: SLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, China National Knowledge Infrastructure, and Wanfang database were summarized. Predicted configurations of wild type (WT) and mutant proteins were achieved using the I-TASSER workplace. Six missense mutations (T60M, L215F, D486N, N534K, Q617R, and R928C) were generated by site-directed mutagenesis. 22Na+ uptake experiment was carried out in the Xenopus laevisoocyte expression system. In the study, 35 GS patients and 20 healthy volunteers underwent the thiazide test. Results: T60M, T163M, D486N, R913Q, R928C, and R959frameshift were frequent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS families. The protein's three-dimensional structure was predicted to be altered in all mutations. Compared with WT hNCC, the thiazide-sensitive 22Na+ uptake was significantly diminished for all six mutations: T60M 22 ± 9.2%, R928C 29 ± 12%, L215F 38 ± 14%, N534K 41 ± 15.5%, Q617R 63 ± 22.1%, and D486N 77 ± 20.4%. In thiazide test, the net increase in chloride fractional excretion in 20 healthy controls was significantly higher than GS patients with or without T60M or D486N mutations. Conclusions: Frequent mutations (T60M, D486N, and R928C) and novel mutations (L215F, N534K, and Q617R) lead to protein structure alternation and protein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on the patients.

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???displayArticle.pmcLink??? PMC8859957
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Species referenced: Xenopus laevis
Genes referenced: slc12a3

???displayArticle.disOnts??? Gitelman syndrome
???displayArticle.omims??? GITELMAN SYNDROME; GTLMNS

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	Figure 1. Number of different mutation types found in Chinese GS patients (A) and the number of mutant sites detected in each patient (B). (A) A total of 155 SLC12A3 gene mutations were detected in 338 Chinese GS patients from 310 unrelated families, including 112 missense mutations (72.3%), 9 nonsense mutations (5.8%), 11 splicing mutations (7.1%), 16 small deletions (10.3%), 3 small insertions (1.9%), 3 small indels (1.9%), and 1 gross deletion (0.6%). (B) Five mutant sites were detected in 1 patient, 4 mutant sites were found in 5 patients, 3 mutant sites were carried by 27 patients, 2 mutant sites (compound heterozygous, CH) were detected in 173 patients, 2 mutant sites (homozygous, Homo) were found in 56 patients, and single heterozygous mutation was carried by 76 patients.
	Figure 2. Frequency and distribution of the 155-detected mutations and 591-mutated alleles. A total of 338 Chinese patients from 310 unrelated families were diagnosed by SLC12A3 gene sequencing, and 155 mutations and 591 mutated alleles were detected in these families. T60M, T163M, D486N, R913Q, R928C, and R959frameshift were the most frequent mutations (mutated allele frequency greater than 3%). On the horizontal axis, each bar represents one mutation (there is no relationship with the actual position in the exon). The dotted line corresponds to an allele frequency of 3% (n = 17.73).
	Figure 3. Predicted configuration of WT and six frequent mutant hNCC proteins. Figures A–F are configurations of WT hNCC proteins with local amplification of T60 (A), T163 (B), D486 (C), R913 (D), R928 (E), and R959 (F). Figure A′–F′ are the configurations of corresponding mutant hNCC proteins with local amplification of M60 (A′), M163 (B′), N486 (C′), Q913 (D′), C928 (E′), and 959frameshift (F′).
	Figure 4. DNA sequence results of six mutated hNCC-pGEM T vectors. The DNA sequence results of six mutated hNCC-pGEM T vectors (T60M, L215F, D486N, N534K, Q617R, and R928C). The mutations’ sequence results are listed at the left column and the corresponding sites of WT are presented at the right column. The sites of mutations are denoted by red arrows.
	Figure 5. Location of the six studied SLC12A3 mutations in the predicted hNCC protein and the result of 22Na+ uptake experiment. (A) The schematic topological representation of hNCC consists of the intracellular N- and C-terminal domains and 12 transmembrane segments. We studied the function of the mutations labeled T60M, L215F, D486N, N534K, Q617R, and R928C. (B) Metolazone-sensitive 22Na+ uptake was measured in oocytes injected with H2O, WT (open bars), or mutant SLC12A3 cRNAs (black bars). The uptake values are shown as percentages of WT 22Na+ transport (WT was set as 100%). (C) Time course of 22Na+ uptake in WT and hNCC mutant-injected X. laevis oocytes. X. laevisoocytes were microinjected with the following SLC12A3 cRNAs: WT, sham, and mutants T60M, L215F, D486N, N534K, Q617R, and R928C. Data were presented as the mean ± s.e.m. and compared using unpaired t-tests. *P< 0.05 indicates a significant difference compared with WT SLC12A3-injected oocytes. Each data point represents the mean value of 10–15 oocytes.
	Figure 6. The thiazide test result verified hNCC dysfunction in GS patients, while no apparent difference was found among the GS patients with or without T60M or D486N mutation. The net increase in chloride fractional excretion (FECl) undergoing thiazide test in 20 healthy controls (4.46 ± 1.04%), 3 GS patients with T60M mutation (2.07 ± 0.62%), 7 GS patients with D486N mutation (1.13 ± 1.19%), and 25 GS patients without any T60M or D486N mutation (0.92 ± 1.09%). One-way ANOVA was performed to evaluate the differences among three subgroups of GS patients, followed by least significant difference post hoc test for each two subgroups. The differences in thiazide test between healthy volunteers and each subgroup of GS patients were compared using unpaired t-tests.

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